Project summary During cell division, duplicated chromosomes are segregated by an exquisite molecular machine, the mitotic spindle. Our goal is to uncover how this machine operates by reconstituting spindle activities and applying advanced biophysical tools for manipulating and tracking individual molecules. We focus on the components most central to spindle function, kinetochores, microtubules, and spindle poles. Kinetochores drive chromosome movements by maintaining persistent, load-bearing attachments to microtubule tips, even as the tips assemble and disassemble under their grip. Kinetochores also somehow sense when they are erroneously attached and, if so, they detach and generate diffusible ?wait? signals to delay anaphase until proper attachments are made. Spindle microtubules are organized into a bipolar configuration by the spindle poles, which also must sustain forces to support chromosome movements and spindle assembly. In past work, we have developed motility assays where native kinetochores or recombinant kinetochore subcomplexes are attached to individual dynamic microtubules. Like kinetochores in vivo, the isolated kinetochore particles remain tip-bound even as the microtubule tips assemble and disassemble ? a behavior we call ?tip-coupling?. We have also reconstituted attachments between microtubules and spindle pole bodies, the yeast counterparts of centrosomes, and made the first measurements of their mechanical strength. Altogether our reconstitutions have enabled us to make key discoveries in major areas of spindle function. By expanding our approach, we can now attack the essence of many complex, long-standing problems in mitosis, in direct ways that would be impossible in living cells. Over the next five years, we will focus on several important questions: (1) How do kinetochores spontaneously self-assemble from their component parts? (2) How are forces transmitted from the outer microtubule-binding interface through the middle of the kinetochore and ultimately to the centromeric DNA? (3) How are dynamic behaviors at kinetochores and spindle poles affected by the forces they experience? (4) How do kinetochores avoid making erroneous attachments? (5) How do unattached or erroneously attached kinetochores generate ?wait? signals to delay the cell cycle? Our work will continue to use the advanced, feedback-controlled laser traps that we pioneered for measuring kinetochore movement and spindle pole mechanics. In addition, newly developed fluorescence techniques will allow us to observe kinetochore assembly at the single molecule level and to monitor dynamic structural changes within individual kinetochores. By combining laser trapping with fluorescence we will test directly how changes in the composition and architecture of kinetochores and spindle poles affect their function.